1. Field of the Invention
This invention relates to variant aequorin genes and a process for producing variant aequorin proteins. More particularly it relates to variant aequorin genes prepared according to a site-specific mutagenesis method using a synthetic oligonucleotide, and a process for producing the above-mentioned proteins by the use of the above-mentioned genes.
2. Description of the Related Art
Aequorin existent in nature is the so-called photoprotein separated from photogenic Aequorea living in the sea, followed by separation and purification, and has been known as a biologically active substance in living body having a high utility value. Namely, since aequorin emits light by way of metal ions such as Ca.sup.2+, Sr.sup.2+, etc., it is utilized as a reagent for detecting trace Ca.sup.2+ (10.sup.-9 M), and in particular, it has been confirmed to be effective for measuring intercellular Ca.sup.2+. However, its production quantity is extremely small so that it is the present status that the quantity is insufficient even as an agent for research.
Thus, firstly the present inventors separated cDNA gene from photogenic Aequorea, identified it and referred to it as pAQ440 (Japanese patent application laid-open No. Sho 61-135586/1986). Further, we succeeded in producing aequorin protein inside Escherichia coli by means of recombinant DNA technique (Japanese patent application No. Sho 60-280259/1985), and also disclosed that it is possible to detect metal ions such as Ca.sup.2+ by making use of this aequorin protein (Japanese patent application No. Sho 61-103849/1986).
However, as to its photogenic mechanism, many unclarified points are still present. Elucidation of the photogenic mechanism of aequorin protein and its correct understanding will extend a possibility of concrete applications of aequorin protein. More particularly, understanding of aequorin as a functional protein having a utility in the aspect of structure and function of protein will be linked to elucidation of the photogenic mechanism of aequorin and also will have a profound meaning in the aspect of protein engineering and further a commercial utilization value.
In view of the technical situation relative to aequorin protein, the present inventors have prepared variants of natural type aequorin gene (pAQ440) by means of recombinant DNA technique, and have succeeded in producing variant aequorin genes inside Escherichia coli by making use of the above genes. Further, by comparing the structure and function of these variant aequorin genes with those of pAQ440, it has become possible to more profoundly analyze the photogenic mechanism of the latter pAQ440.
As apparent from the foregoing, the object of the present invention is to provide many kinds of specified variant aequorin genes useful for making the above-mentioned analysis possible, and a process for producing variant aequorin proteins by the use of the above variant aequorin genes.
Further, as described above, as to the photogenic mechanism, the present inventors have analyzed the structure and function of the aequorin gene according to the site-specific mutagenesis method (Japanese patent application Nos. Sho 61-245108/1986 and Sho 61-245109/1986).
However, during the regeneration process of aequorin wherein aequorin which is light-emissive due to calcium is reconstructed in the presence of apoaequorin, coelenterazine as a substrate, molecular form oxygen and 2-mercaptoethanol as a reducing agent, it has been known that 2-mercaptoethanol is necessary to be in a high concentration. The reason why 2-mercaptoethanol is required is unclear, but a possibility of converting the --S--S--bond of aequorin protein(apoaequorin) into --SH,HS--is suggested.
Thus, it is very meaningful to produce variant aequorin proteins which do not require the presence of 2-mercaptoethanol as a reducing agent at the time of regeneration of aequorin by means of recombinant DNA technique, and this will be linked to elucidation of the regeneration mechanism of aequorin and further it will have a profound meaning in the aspect of protein engineering and also a utilization value in the scientific and commercial aspect.
In view of the above-mentioned technical situation of aequorin protein, the present inventors have prepared variants of natural type aequorin gene (pAQ440) by means of recombinant DNA technique, and have succeeded in producing variant aequorin genes inside Escherichia coli by making use of these genes.
Further, it has become possible to produce apoaequorin from which regeneration of aequorin is possible without needing the presence of 2-mercaptoethanol, using variant aequorin genes of the present invention as described later. The variant aequorin genes could have been obtained by converting G of TGC as a base arrangement which can form cysteine residual group on the aequorin gene, into C, to thereby exchange the serine residual group into the cysteine residual group in apoaequorin molecule.